Frank M. Graziano

Olopatadine inhibits TNFα release from human conjunctival mast cells

Background Tumor necrosis factor-α (TNFα) release likely plays a crucial role in allergic ocular inflammation via increasing ICAM-1 on epithelial cells and triggering other proinflammatory events. The immediate and prolonged release of TNFα from human conjunctival mast cells in response to allergen challenge is potentially an important target for therapeutic intervention, yet the effect of ocular anti-allergic agents on this process has not been examined. Olopatadine (Patanol) is a clinically effective dual-action ophthalmic anti-allergic agent that has been shown to inhibit mast cell histamine, tryptase, and PGD 2 release in vitro and promote decreased H 1 receptor binding activity in vitro and functional H 1 receptor antagonism in vivo. Objective To investigate the effect of olopatadine on TNFα release from anti-IgE antibody challenged purified human conjunctival mast cells. Methods Human conjunctival mast cells were purified (>95%) from cadaveric tissues using a procedure combining enzymatic digestion and Percoll gradient centrifugation. These cells were incubated with olopatadine for 30 minutes then challenged with anti-IgE antibody for 90 minutes. Supernatants were analyzed for TNFα. Results Purified human conjunctival mast cells responded to anti-IgE antibody challenge with TNFα release in a concentration dependent manner (optimum concentration was 10 μg/mL). Olopatadine pre-incubation resulted in a dose-dependent decrease in anti-IgE antibody mediated TNFα release (IC 50 = 13.1 μM). At a concentration of 3 mM olopatadine reduced TNFα release to the level of unchallenged controls. Conclusion Olopatadine inhibited anti-IgE antibody-mediated release of TNFα from human conjunctival mast cells. This effect could contribute to the long duration of anti-allergic activity reported for the drug.

Autologous hematopoietic stem cell transplantation in refractory rheumatoid arthritis: Sustained response in two of four patients

OBJECTIVE To investigate the safety and efficacy of immune ablation with subsequent autologous hematopoietic stem cell transplantation (HSCT) in severe rheumatoid arthritis (RA). METHODS Four patients with refractory RA and poor prognostic indicators were treated. Stem cells were collected and lymphocytes were depleted by 2.3-4.0 logs. The conditioning regimen included cyclophosphamide (200 mg/kg), antithymocyte globulin (90 mg/kg), and, for 1 patient, total body irradiation (TBI) with 400 cGy. Improvement was evaluated according to the American College of Rheumatology (ACR) preliminary definition of improvement in RA (ACR 20), and also according to the ACR 50 and ACR 70 criteria. RESULTS HSCT was well tolerated. Three patients fulfilled the ACR 70 criteria at 1 month and 3 months post-HSCT. One patient did not fulfill the ACR 20 criteria because of persistent joint tenderness, despite improvement of the joint swelling. At 6 months post-HSCT, 1 patient fulfilled the ACR 70 criteria and 1 fulfilled the ACR 50 criteria, and these 2 patients fulfilled the ACR 70 criteria at 9 months post-HSCT. The other 2 patients (including the patient who received TBI) did not meet the ACR 20 criteria at 6 months and 9 months post-HSCT. The only patient with followup of >9 months fulfilled the ACR 70 criteria at 20 months post-HSCT. CONCLUSION In this series, autologous HSCT was safe and effective in inducing major clinical response and maintained significant benefit for 2 patients at 9 months and 20 months posttreatment, respectively. Sustained response did not occur for 2 of 4 patients. A regimen dose-response effect may exist, but the addition of TBI did not prevent disease relapse for 1 of the patients. More aggressive T cell depletion of the autograft, use of a myeloablative regimen, or use of an allograft may be necessary to decrease relapse rates.

The presence of hypodense eosinophils and diminished chemiluminescence response in asthma

Although peripheral blood eosinophilia is a prominent feature of asthma, the contribution of eosinophils to asthma has yet to be fully comprehended. Furthermore, study of isolated eosinophil function in asthma has been complicated by difficult purification methods and, now, the presence of hypodense eosinophils. In our study, eosinophils were isolated from normal subjects and patients with asthma. Two principal evaluations were performed: (1) a comparison of the density-gradient profiles on peripheral blood leukocytes from normal subjects and patients with asthma and (2) a comparison of the chemiluminescence (CL) response with normal dense eosinophils from these two study groups. Granulocyte preparations were initially isolated from Ficoll-Hypaque gradients and were then applied to a continuous Percoll density gradient. In asthma, 40.8 +/- 5.8% of the peripheral blood eosinophils were hypodense (defined as a density less than 1.081 gm/ml), whereas normal subjects had only 9.1 +/- 1.9% of this subpopulation (p less than 0.01). Functional assessment of purified (greater than 90%) normal dense eosinophils was made by measurement of CL to opsonized zymosan particles and the soluble stimulus phorbol myristate acetate. In asthma, eosinophil CL to zymosan, but not phorbol myristate acetate, was significantly less. Differences in eosinophil CL between normal subjects and subjects with asthma did not correlate with the severity of airway obstruction or the peripheral blood eosinophil count. The reasons for the appearance of hypodense eosinophils and diminished metabolic activity in asthma are not established but raise the possibility that their presence represents previous eosinophil activation.

Multicenter trial of octreotide in patients with refractory acquired immunodeficiency syndrome-associated diarrhea

BACKGROUND/AIMS Diarrhea is a significant problem in patients with acquired immunodeficiency syndrome (AIDS). The aim of this study was to determine octreotide effectiveness in refractory AIDS-associated diarrhea. METHODS In a 3-week protocol, 129 patients with a stool weight of > 500 g/day despite standard antidiarrheal therapy were randomized to receive octreotide or placebo (3:2 ratio). Octreotide dose was increased 100 micrograms weekly to a maximum of 300 micrograms three times a day based on weekly 72-hour stool collections. Subsequently, patients received open-label octreotide at doses of up to 500 micrograms three times a day. RESULTS A 30% decrease in stool weight defined response. After 3 weeks, 48% of octreotide- and 39% of placebo-treated patients had responded (P = 0.43). At 300 micrograms three times a day, 50% of octreotide- and 30.1% of placebo-treated patients responded (P = 0.12). At a baseline stool weight of 1000-2000 g/day, 57% of octreotide- and 25% of placebo-treated patients responded (P = 0.06). Response rates based on CD4 counts, diarrhea duration, body weight, human immunodeficiency virus risk factor, and presence or absence of pathogens showed no benefit of octreotide. Adverse events were more frequent in the octreotide-treated group. CONCLUSION In the doses studied, octreotide was not more effective than placebo in patients with refractory AIDS-associated diarrhea. This lack of effectiveness may be attributable to inadequate sample size, doses, and duration of study treatment.

Adrenal insufficiency in the antiphospholipidantibody syndrome

Abstract Objective: Adrenal insufficiency (AI) is a rare complication of the antiphospholipid antibody syndrome (APS). The objective of this report is to describe a case and review the published literature to enhance recognition of this potentially fatal disorder by emphasizing its course, diagnosis, and cause. Data Sources: A bibliographic database with the indexing terms adrenal insufficiency, adrenal hemorrhage, adrenal thrombosis, APS, systemic lupus erythematosus, with the constraints of human subjects only, was used. Study Selection: All 27 reports meeting the indexing terms were selected for review. Data Extraction: The specific criteria used for data extraction articles includedcourse of the disease, causation, clinical and laboratory diagnostic criteria, and therapeutic intervention. Data Synthesis: Our patient is a previously health woman who developed a respiratory tract infection, followed by a prolonged illness with fever, hypotension, nausea, depression, and venous thromboses. She was found to have AI and APS that was alleviated with hydrocortisone and anticoagulation. Initially, her adrenal glands were normal on CT scan but subsequently became enlarged and later atrophic. Of the 27 previous case reports, a majority had thromboses and typical clinical and laboratory manifestations of Al. Hemorrhagic infarction of the adrenal gland appears to be the mechanism for AI in the APS. IgG and IgM anticardiolipin antibodies are most commonly reported in association with Al in APS. Conclusions: The hypercoagulable state in the APS may lead to adrenal vein thrombosis and subsequently to hemorrhagic necrosis of the adrenal gland. This complication of APS is important to recognize because it may be fatal if untreated.

Cytokines, chemokines, RANTES, and eotaxin.

Over the past several years, a number of cytokines with chemoattractive properties (chemokines) have been identified. These low molecular weight molecules have been shown to be important leukocyte chemical attractants to sites of inflammation and infection. Chemokines act on leukocytes through selective receptors and are now known to function also in leukocyte maturation, trafficking, and homing of these cells. RANTES and eotaxin (among other chemokines) are important chemoattractants for eosinophils. Since eosinophils seem to play a critical role in the production of allergic inflammation, an understanding of the mechanism of action of these chemokines may lead to new therapies for asthma and other allergic processes.

Olopatadine inhibits anti-immunoglobulin E- stimulated conjunctival mast cell upregulation of ICAM-1 expression on conjunctival epithelial cells

BACKGROUND Olopatadine is a clinically effective dual-action (antihistamine/mast cell stabilizer) ophthalmic antiallergic agent. We have previously demonstrated that olopatadine inhibits tumor necrosis factor alpha (TNF-alpha) release from purified human conjunctival mast cells and that supernates from stimulated mast cells upregulate intercellular adhesion molecule 1 (ICAM-1) expression on epithelial cells via TNF-alpha. OBJECTIVE To investigate the effect of olopatadine on the TNF-alpha-mediated mast cell upregulation of ICAM-1 expression on conjunctival epithelial cells. METHODS Human conjunctival mast cells and epithelial cells were purified (>95%) from cadaveric tissue. Conjunctival mast cells were preincubated with three doses (30, 300, or 3,000 microM) of olopatadine or buffer alone for 30 minutes followed by 90-minute challenge with anti-immunoglobulin E (10 microg/mL). The resulting supernates were incubated with conjunctival epithelial cell monolayers for 24 hours along with the following treatments: rTNF-alpha, mast cell supernate + anti-TNF-alpha, recombinant (r)TNF-alpha + anti-TNF-alpha, the three doses of olopatadine, olopatadine supernates, olopatadine supernates + rTNF-alpha. ICAM-1 expression was measured using flow cytometry. RESULTS Anti-IgE-stimulated human conjunctival mast cell supernates upregulated human conjunctival epithelial cell ICAM-1 expression to the same extent as rTNF-alpha. ICAM-1 upregulation could be completely blocked with anti-TNF-alpha. Preincubation of conjunctival mast cells with olopatadine significantly blocked the ability of supernates to upregulate ICAM-1 on conjunctival epithelial cells. ICAM-1 expression could be restored by adding rTNF-alpha to the olopatadine-preincubated mast cell supernates. CONCLUSIONS Olopatadine is able to significantly decrease the anti-immunoglobulin E mast cell supernate-mediated upregulation of ICAM-1 on human conjunctival epithelial cells in vitro. This seems to be mediated through an effect on a TNF-alpha-specific mechanism.

Enhanced eosinophil luminol-dependent chemiluminescence in allergic rhinitis.

Eosinophils were isolated from patients with seasonal allergic rhinitis, and their functional activity was evaluated by their luminol-dependent chemiluminescence (CL) response to opsonized zymosan and phorbol 12-myristate 13-acetate (PMA). We found that eosinophils from patients with allergic rhinitis produced a significantly greater CL response to opsonized zymosan than did normal cells. Eosinophils from both subjects with allergic rhinitis and control subjects were isolated to a purity of 95% and elicited peak values of 1,101, 901 +/- 133,708 cpm/5 X 10(5) cells (n = 7) and 417,278 +/- 25,910 cpm/5 X 10(5) cells (n = 5), respectively. The enhanced eosinophil CL to zymosan was found at times when the patients were maximally symptomatic with hay fever symptoms to ragweed pollen and again when they were asymptomatic. Eosinophils from these patients with allergic rhinitis also had enhanced CL to PMA (0.01 mcg/ml), but this increased activity was largely limited to times of hay fever symptoms. No correlation was found between enhanced eosinophil CL activity and the number of circulating eosinophils in allergic individuals. In contrast to the increased eosinophil activity, neutrophil CL to zymosan and PMA was similar in allergic and normal individuals throughout the study. These data provide evidence for the existence of enhanced oxidative metabolic function in eosinophils from patients with allergic rhinitis and raise the possibility that this particular activity is important in hay fever.

A guinea pig model for study of bladder mast cell function : histamine release and smooth muscle contraction

To study the function of mast cells in bladder tissue, guinea pigs were sensitized with ovalbumin by intraperitoneal injections, bladder tissue strips were superfused, and tissue contractile force and histamine release were studied. Upon challenge with ovalbumin, bladder tissue contracted 64 +/- 4% (mean +/- S.E.M.) of the maximum carbachol contraction and released 14.1 +/- 1.6% of the total tissue histamine content. Incubation of sensitized bladder tissue with indomethacin led to an increased force and duration of the contraction while incubation with nordihydroguaiaretic acid combined with pyrilamine reduced histamine release and abolished the contraction. Tissue histamine content was significantly higher in the bladder neck than in the dome, and significantly elevated following sensitization. Histochemical studies of bladder tissue demonstrated mast cell degranulation in antigen challenge experiments. In addition, a group of guinea pigs were sensitized to ovalbumin through bladder instillations. With this model, study of the functional characteristics of bladder mast cells and the acute actions of mast cell products on the bladder microenvironment, should now be feasible.

Toll-like receptor 2 expression on human conjunctival epithelial cells: a pathway for Staphylococcus aureus involvement in chronic ocular proinflammatory responses

BACKGROUND Staphylococcus aureus colonization is common in atopic keratoconjunctivitis, potentially activating epithelial cells via toll-like receptor 2 (TLR-2) and the receptor for platelet-activating factor (PAFR). OBJECTIVES To examine human conjunctival epithelial cells for the expression of TLR-2 in vitro and in vivo and to evaluate the role of TLR-2 in S aureus-mediated activation of these cells. METHODS Conjunctival epithelial cells isolated from cadaveric tissues were stimulated with interferon gamma (IFN-gamma) or a commercial S aureus cell wall extract (Staphylococcus aureus-CWE) (with or without anti-TLR-2 blocking antibody or PAFR antagonist) and were analyzed for tumor necrosis factor alpha (TNF-alpha) and interleukin 8 (IL-8) release; surface expression of TLR-2, intercellular adhesion molecule-1, HLA, and CD14; and TLR-2 messenger RNA expression. Ocular surface cells collected via impression cytology were examined for TLR-2 expression via flow cytometry. RESULTS Expression of TLR-2 was up-regulated on conjunctival epithelial cells by IFN-gamma and Staphylococcus aureus-CWE. Expression of TLR-2 messenger RNA was increased by IFN-gamma. Staphylococcus aureus-CWE up-regulated intercellular adhesion molecule 1, HLA, and CD14 expression and increased TNF-alpha and IL-8 release in a dose-dependent manner. Anti-TLR-2 significantly inhibited TNF-alpha release, whereas PAFR antagonist significantly inhibited IL-8 release. Toll-like receptor 2 was expressed on conjunctival epithelial cells from 4 of 5 patients with atopic keratoconjunctivitis, 3 of 5 with seasonal allergies, and 0 of 3 without allergies. CONCLUSIONS Conjunctival epithelial cells express TLR-2 and may play an active role in the chronic ocular inflammatory response to S aureus through pathways that involve TLR-2 and PAFR.